Dataset Information

[Construction and expression of nm23-H1 gene with different enzyme activities and resistant specific shRNA in eukaryotic expression vector].

ABSTRACT:

Background and objective

It has been proven that nm23-H1 gene was a tumor metastatic suppressor gene. However, it’s molecular mechanism of suppressing metastasis remains unexplored. There is a closely relationship between the abnormality of stucturs and functions of nm23-H1 gene, and cancer invasion and metastasis. We have constructed the vector with nm23-H1-shRNA and the vector with nm23-H1cDNA resistant to the specific shRNA. So, we plan to construct shRNA-resistant eukaryotic expression vector of nm23-H1 gene by site-directed mutagenesis, rescue experiment was performed to verify the nm23-H1 gene expression, and to provide basement for studying the biochemical mechanisms of nm23-H1 gene.

Methods

Site-directed mutagenesis of nm23-H1 gene was performed by overlap extension PCR method. Pure plasmid containing gene of nm23-H1 (shRNA-resistant) was prepared. The desired five mutations were constructed and cloned into the eukaryotic vector pcDNA3.1Hygro(+). The human lung adenocarcinoma cell A549/nm23-H1-shRNA (stable nm23-H1 gene silencing) was transfected with the five mutants, and the expression of the mutant proteins was determined by Western blot.

Results

Five eukaryotic expression vectors (shRNA-resistant) of nm23-H1, nm23-H1S44A, nm23-H1(P96S), nm23-H1(H118F), nm23-H1(S120G), nm23-H1(P96S-S120G), were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the genes were completely concordant with experiment design. The expression of nm23-H1 mutant proteins was verified by Western blot.

Conclusion

Five eukaryotic expression vectors (shRNA-resistant) of nm23-H1 gene were successfully constructed, and the mutant proteins were verified. The site-directed mutagenesis technical of overlap extension PCR is a efficient, simple and economical method.

SUBMITTER: Lu Z

PROVIDER: S-EPMC6019366 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Publications

[Construction and expression of nm23-H1 gene with different enzyme activities and resistant specific shRNA in eukaryotic expression vector].

Lu Zhansheng Z Guo Lili L Li Lin L Wu Zhihao Z Zhou Qinghua Q

Zhongguo fei ai za zhi = Chinese journal of lung cancer 20140301 3

<h4>Background and objective</h4>It has been proven that nm23-H1 gene was a tumor metastatic suppressor gene. However, it’s molecular mechanism of suppressing metastasis remains unexplored. There is a closely relationship between the abnormality of stucturs and functions of nm23-H1 gene, and cancer invasion and metastasis. We have constructed the vector with nm23-H1-shRNA and the vector with nm23-H1cDNA resistant to the specific shRNA. So, we plan to construct shRNA-resistant eukaryotic expressi ...[more]

PMID: 24667253

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Project description:Influenza (flu) pandemics have exhibited a great threat to human health throughout history. With the emergence of drug-resistant strains of influenza A virus (IAV), it is necessary to look for new agents for treatment and transmission prevention of the flu. Defensins are small (2-6 kDa) cationic peptides known for their broad-spectrum antimicrobial activity. Beta-defensins (β-defensins) are mainly produced by barrier epithelial cells and play an important role in attacking microbe invasion by epithelium. In this study, we focused on the anti-influenza A virus activity of mouse β-defensin 1 (mBD1) and β defensin-3 (mBD3) by synthesizing their fusion peptide with standard recombinant methods. The eukaryotic expression vectors pcDNA3.1(+)/mBD1-mBD3 were constructed successfully by overlap-PCR and transfected into Madin-Darby canine kidney (MDCK) cells. The MDCK cells transfected by pcDNA3.1(+)/mBD1-mBD3 were obtained by G₄₁₈ screening, and the mBD1-mBD3 stable expression pattern was confirmed in MDCK cells by RT-PCR and immunofluorescence assay. The acquired stable transfected MDCK cells were infected with IAV (A/PR/8/34, H1N1, 0.1 MOI) subsequently and the virus titers in cell culture supernatants were analyzed by TCID5₅₀ 72 h later. The TCID₅₀ titer of the experimental group was clearly lower than that of the control group (p < 0.001). Furthermore, BALB/C mice were injected with liposome-encapsulated pcDNA3.1(+)/mBD1-mBD3 through muscle and then challenged with the A/PR/8/34 virus. Results showed the survival rate of 100% and lung index inhibitory rate of 32.6% in pcDNA3.1(+)/mBD1-mBD3group; the TCID₅₀ titer of lung homogenates was clearly lower than that of the control group (p < 0.001). This study demonstrates that mBD1-mBD3 expressed by the recombinant plasmid pcDNA3.1(+)/mBD1-mBD3 could inhibit influenza A virus replication both in vitro and in vivo. These observations suggested that the recombinant mBD1-mBD3 might be developed into an agent for influenza prevention and treatment.

Dataset Information

[Construction and expression of nm23-H1 gene with different enzyme activities and resistant specific shRNA in eukaryotic expression vector].

Background and objective

Methods

Results

Conclusion

Publications

[Construction and expression of nm23-H1 gene with different enzyme activities and resistant specific shRNA in eukaryotic expression vector].

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